Griseoviridin is a group A streptogramin natural product from Streptomyces with broad-spectrum antibacterial activity. A hybrid polyketide-nonribosomal peptide, it comprises a 23-membered macrocycle, an embedded oxazole motif, and a macrolactone with a unique ene-thiol linkage. Recent analysis of the griseoviridin biosynthetic gene cluster implicated SgvP, a cytochrome P450 monooxygenase, in late-stage installation of the critical C-S bond. While genetic and crystallographic experiments provided indirect evidence to support this hypothesis, the exact function of SgvP has never been confirmed biochemically. Herein, we report a convergent total synthesis of pre-griseoviridin, the putative substrate of P450 SgvP and precursor to griseoviridin. Our strategy features concise and rapid assembly of two fragments joined via sequential peptide coupling and Stille macrocyclization. Access to pre-griseoviridin then enabled in vitro validation of SgvP as the C-S bond-forming P450 during griseoviridin biosynthesis, culminating in a nine-step chemoenzymatic synthesis of griseoviridin.